Methods of detecting mutations associated with ataxia-ocular apraxia 2 (AOA2)

ABSTRACT

Methods of identifying polymorphisms associated with ataxia-ocular apraxia 2 (AOA2), are described. The polymorphisms associated with AOA2 include specific mutations in the senataxin (SETX) gene. Also described are methods of diagnosis of AOA2, as well as methods of assessing an individual for carrier status for AOA2.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/762,815, filed on Jan. 27, 2006. The entire teachings of the aboveapplication are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Ataxia-ocular apraxia 2 (AOA2), or ataxia with oculomotor apraxia type2, is a recently identified autosomal recessive cerebellar ataxia (LeBer, I. et al., Curr. Neurol. Neurosci. Rep. 2005, 5(5):411-7).Initially associated with a locus at 9q34 (Bomont, P. et al., Eur. J.Hum. Genet. 2000, 8:986-990; Nemeth, A. H. et al., Am. J. Hum. Genet.2000, 67:1320-1326; Le Ber, I. et al., Brain 2004, 127: 759-767; Izatt,L. et al., J. Neurol. 2004, 251:805-812; Mahajnah, M. et al., J. ChildNeurol. 2005, 20(5):523-525), AOA2 has now been linked to mutations inthe senataxin (SETX) gene at that locus (Moreira, M-C. et al., Nat.Genet. 2004, 36(3):225-227; Duquette, A. et al., Ann. Neurol. 2005,57:408-414). Certain mutations in the 9q34 locus, including mutations inthe SETX gene, have also been associated with an autosomal dominantjuvenile amyotrophic lateral sclerosis (ALS4) (Chance, P. F. et al., Am.J. Hum. Genet. 1998, 62:633-640; Rabin, B. A. et al., Brain 1999,122:1539-1550; Blair, I. P. et al., Neurogenetics 2000, 3:1-6; Chen,Y.-Z. et al., Am. J. Hum. Genet. 2004, 74:1128-1135). In addition, AOA2shares some similarities with other autosomal recessive cerebellarataxias (ARCAs), including ataxia-telangiectasia (A-T) (Chun, H. H. AndR. A. Gatti, DNA Repair, 2004, 3:1187-1196) and ataxia with oculomotorapraxia type 1 (AOA1) (Le Ber, I. et al., Brain 2003, 126:2761-2772). Aneed remains for means to distinguish AOA2 from other diseases, andparticularly from ALS4 and AOA1.

SUMMARY OF THE INVENTION

The present invention is drawn to methods of assessing an individual forthe presence or absence of a genetic polymorphism associated withataxia-ocular apraxia 2 (AOA2). In the methods of the invention, a testsample from the individual is assessed for the presence of at least onemutation of interest in the senataxin (SETX) gene. Assessing the testsample can be performed by standard methods that may includeamplification of all or a fragment of the senataxin gene, and/or directsequence analysis. The test sample comprises nucleic acids, such asgenomic DNA (e.g., genomic DNA comprising chromosome 9 or a fragmentthereof comprising 9q34). The mutation of interest is selected from thegroup consisting of: a single base insertion of T between nucleotides479-480; a 4 base deletion of nucleotides 4633-4636; a 2 base deletionof nucleotides 6114-6115; a single base transition C→T at nucleotide6292; a 4 base deletion of nucleotides 369-372; a 2 base insertion of ATbetween nucleotides 2747-2748; a single base transition C→T atnucleotide 4234; a single base transition C→T at nucleotide 4816; a 6base deletion of nucleotides 4873-4878 accompanied by an insertion of GGat the same location; a single base insertion of G between nucleotides4891-4892; a 2 base insertion of CA between nucleotides 5301-5302; a 4base deletion of nucleotides 5308-5311; a 2 base deletion of nucleotides5591-5592; a single base deletion of nucleotide 5958; a single baseinsertion of A between nucleotides 6422-6423; and a four base deletionof nucleotides 6848-6851. The presence of at least one of thesemutations of interest is indicative of the presence of a geneticpolymorphism associated with ataxia-ocular apraxia 2.

The methods of the invention additionally include methods of diagnosingataxia-ocular apraxia 2 (AOA2) in an individual, by assessing a testsample from the individual for the presence of at least one mutation ofinterest, as described above, in a first allele of the senataxin gene ofthe individual. The presence of a mutation of interest in the firstallele of the senataxin gene is indicative of ataxia-ocular apraxia 2 ifat least one mutation associated with ataxia-ocular apraxia 2 is alsopresent in the second allele of the senataxin gene. In certainembodiments, both the first and second alleles of the SETX may compriseat least one of the mutations of interest.

The methods of the invention further pertain to methods of assessing anindividual for carrier status for ataxia-ocular apraxia 2, by assessinga test sample from the individual for the presence of a mutation ofinterest, as described above, in first and second alleles of thesenataxin gene of the individual. The presence of the mutation ofinterest in the first allele of the senataxin gene, and the absence ofany mutation associated with ataxia-ocular apraxia 2 in the secondallele of the senataxin gene, is indicative of carrier status forataxia-ocular apraxia 2.

The invention further pertains to kits useful in the methods of theinvention.

The methods of the invention provide simple means to distinguishataxia-ocular apraxia 2 from other cerebellar ataxias, as well as toidentify those who are affected with the disease or who are carriers forthe disease.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of assessing individuals for thepresence or absence of a genetic polymorphism associated withataxia-ocular apraxia 2 (AOA2), as well as methods of diagnosing AOA2 inan individual, and methods of assessing an individual for carrier statusfor AOA2. As described herein, Applicant has identified certainmutations of interest in the senataxin gene (SETX gene) that areassociated with AOA2. The mutations in the SETX gene described hereinare alterations (e.g., deletions, insertions, or transitions) in thenucleic acid sequence of the SETX gene. The position of the mutations inthe sequence of SETX are numbered in relation to the mRNA or cDNAsequence: that is, the numbered position of an altered nucleotide is thenumber of that nucleotide in the mRNA or cDNA sequence. The mRNAsequence associated with the SETX gene is set forth in GenBank accessionnumber AY362728, as updated on Mar. 12, 2004 (also shown in SEQ IDNO:1). The mutations of interest include the following alterations: asingle base insertion of T between nucleotides 479-480; a 4 basedeletion of nucleotides 4633-4636; a 2 base deletion of nucleotides6114-6115; a single base transition C→T at nucleotide 6292; a 4 basedeletion of nucleotides 369-372; a 2 base insertion of AT betweennucleotides 2747-2748; a single base transition C→T at nucleotide 4234;a single base transition C→T at nucleotide 4816; a 6 base deletion ofnucleotides 4873-4878 accompanied by an insertion of GG at the samelocation; a single base insertion of G between nucleotides 4891-4892; a2 base insertion of CA between nucleotides 5301-5302; a 4 base deletionof nucleotides 5308-5311; a 2 base deletion of nucleotides 5591-5592; asingle base deletion of nucleotide 5958; a single base insertion of Abetween nucleotides 6422-6423; and a four base deletion of nucleotides6848-6851.

In the methods of the invention, a test sample from an individual isassessed for the presence of one or more of these particular mutationsin the SETX gene (herein also referred to as the “polymorphisms ofinterest” or “polymorphisms associated with AOA2”). The individual is ahuman individual, and may be of any race and any age, including fetus,infant, juvenile, adolescent, and adult. Representative individualsinclude those who have not previously been diagnosed as having AOA2 oras being a carrier for AOA2, as well as those who have been determinedto be at risk for having AOA2 or for being a carrier for AOA2, and thosewho have been initially diagnosed as being affected by AOA2, whereconfirming information is desired.

The test sample is a sample containing nucleic acids comprising the SETXgene or a fragment of the SETX gene, SETX mRNA or a fragment of SETXmRNA, SETX cDNA or a fragment of SETX cDNA, from the individual. Theterm, “fragment,” as used herein, indicates that the portion of thegene, mRNA or cDNA is a polynucleotide of a length that is sufficient toidentify it as a fragment of SETX: in a representative embodiment, afragment comprises one or more exons of the SETX gene; in anotherrepresentative embodiment, a fragment comprises part of an exon of theSETX gene. The fragment can also include intron/exon junction(s) of theSETX gene.

The test sample is prepared from a biological sample from theindividual. The biological sample can be a sample from any source whichcontains genomic DNA (e.g., chromosomal nucleic acids) or RNA, such as ablood sample, sample of amniotic fluid, sample of cerebrospinal fluid,or tissue sample from skin, muscle, buccal or conjunctival mucosa,placenta, gastrointestinal tract or other organs. A biological sample ofnucleic acid from fetal cells or tissue can be obtained by appropriatemethods, such as by amniocentesis or chorionic villus sampling (director cultured). In certain embodiments, a biological sample containinggenomic DNA comprising chromosome 9 or a fragment thereof (e.g., afragment comprising 9q34, or a fragment comprising one or more exons ofthe SETX gene) is used. A biological sample can be used as the testsample; alternatively, a biological sample can be processed to enhanceaccess to nucleic acids, or copies of nucleic acids (e.g., copies ofnucleic acids comprising the SETX gene), and the processed biologicalsample can then be used as the test sample. For example, in oneembodiment, cDNA is prepared from a biological sample comprising mRNA,for use in the methods. Alternatively or in addition, if desired, anamplification method can be used to amplify nucleic acids comprising allor a fragment of the SETX gene in a biological sample, for use as thetest sample in the assessment for the presence or absence of apolymorphism of interest. For example, in a representative embodiment,each of the exons of the SETX gene can be amplified.

The test sample is assessed to determine whether one or more mutationsof interest in the SETX gene (polymorphisms of interest) are present inthe SETX gene of the individual (e.g., in first and second alleles ofthe SETX gene). In general, detecting a polymorphism of interest may becarried out by determining the presence or absence of nucleic acidscontaining the polymorphism of interest in the test sample.

In a first method, hybridization methods, such as Southern analysis,Northern analysis, or in situ hybridizations, can be used (see CurrentProtocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley &Sons, including all supplements). For example, the presence of thepolymorphism of interest can be indicated by hybridization of nucleicacid in the genomic DNA, RNA, or cDNA to a nucleic acid probe. A“nucleic acid probe”, as used herein, can be a DNA probe or an RNAprobe; the nucleic acid probe can contain at least one polymorphism ofinterest, as described herein. The probe can be, for example, the gene,a gene fragment (e.g., one or more exons), a vector comprising the gene,a probe or primer, etc.

To detect one or more of the polymorphisms of interest, a hybridizationsample is formed by contacting the test sample with at least one nucleicacid probe. A preferred probe for detecting mRNA or genomic DNA is alabeled nucleic acid probe capable of hybridizing to mRNA or genomic DNAof the SETX gene. The nucleic acid probe can be, for example, afull-length nucleic acid molecule, or a portion thereof, such as anoligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides inlength and sufficient to specifically hybridize under stringentconditions to appropriate mRNA or genomic DNA. The hybridization sampleis maintained under conditions which are sufficient to allow specifichybridization of the nucleic acid probe to mRNA or genomic DNA of theSETX gene. “Specific hybridization”, as used herein, indicates exacthybridization (e.g., with no mismatches). Specific hybridization can beperformed under high stringency conditions or moderate stringencyconditions, for example, as described above. In a particularly preferredembodiment, the hybridization conditions for specific hybridization arehigh stringency.

Specific hybridization, if present, is then detected using standardmethods. If specific hybridization occurs between the nucleic acid probeand SETX gene or mRNA in the test sample, the polymorphism that ispresent in the nucleic acid probe is also present in the SETX gene ofthe individual. More than one nucleic acid probe can also be usedconcurrently in this method. Specific hybridization of any one of thenucleic acid probes is indicative of the presence of polymorphism ofinterest, as described herein.

In Northern analysis (see Current Protocols in Molecular Biology,Ausubel, F. et al., eds., John Wiley & Sons, supra), the hybridizationmethods described above are used to identify the presence of apolymorphism of interest. For Northern analysis, a test samplecomprising RNA is prepared from a biological sample from the individualby appropriate means. Specific hybridization of a nucleic acid probe, asdescribed above, to RNA from the individual is indicative of thepresence of a polymorphism of interest, as described herein.

For representative examples of use of nucleic acid probes, see, forexample, U.S. Pat. Nos. 5,288,611 and 4,851,330.

Alternatively, a peptide nucleic acid (PNA) probe can be used instead ofa nucleic acid probe in the hybridization methods described above. PNAis a DNA mimic having a peptide-like, inorganic backbone, such asN-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U)attached to the glycine nitrogen via a methylene carbonyl linker (see,for example, Nielsen, P. E. et al., Bioconjugate Chemistry, 1994, 5,American Chemical Society, p. 1 (1994). The PNA probe can be designed tospecifically hybridize to a SETX gene comprising one-or more of thepolymorphisms of interest described herein. Hybridization of the PNAprobe to a SETX gene is indicative of the presence of the polymorphismof interest.

In another method of the invention, mutation analysis by restrictiondigestion can be used to detect a mutant SETX gene, or an SETX genecontaining a polymorphism(s) of interest, if the mutation orpolymorphism in the SETX gene results in the creation or elimination ofa restriction site. A sample containing genomic DNA from the individualis used. Polymerase chain reaction (PCR) can be used to amplify all or afragment of the SETX gene (and, if necessary, the flanking sequences) inthe sample. RFLP analysis is conducted as described (see CurrentProtocols in Molecular Biology, supra). The digestion pattern of therelevant DNA fragment indicates the presence or absence of polymorphismin the SETX gene.

Direct sequence analysis can also be used to detect specificpolymorphisms of interest in the SETX gene. A sample comprising genomicDNA or RNA is used, and PCR or other appropriate methods can be used toamplify all or a fragment of the SETX gene, and/or its flankingsequences, if desired. The sequence the SETX gene, or a fragment of thegene (e.g., one or more exons), or cDNA, or fragment of the cDNA, ormRNA, or fragment of the mRNA, is determined, using standard methods.The sequence of the gene, gene fragment, cDNA, cDNA fragment, mRNA, ormRNA fragment is compared with the known nucleic acid sequence of theSETX gene, cDNA or mRNA, as appropriate. The presence of a polymorphismof interest can then be identified.

Allele-specific oligonucleotides can also be used to detect the presenceof a polymorphism of interest, through the use of dot-blot hybridizationof amplified oligonucleotides with allele-specific oligonucleotide (ASO)probes (see, for example, Saiki, R. et al., (1986), Nature (London)324:163-166). An “allele-specific oligonucleotide” (also referred toherein as an “allele-specific oligonucleotide probe”) is anoligonucleotide of approximately 10-50 base pairs, preferablyapproximately 15-30 base pairs, that specifically hybridizes to the SETXgene, and that contains a polymorphism of interest as described herein.An allele-specific oligonucleotide probe that is specific for particularpolymorphisms can be prepared, using standard methods (see CurrentProtocols in Molecular Biology, supra). To identify polymorphisms ofinterest, a sample comprising DNA is used. PCR can be used to amplifyall or a fragment of the SETX gene, and its flanking sequences. The DNAcontaining the amplified SETX gene (or fragment of the gene) isdot-blotted, using standard methods (see Current Protocols in MolecularBiology, supra), and the blot is contacted with the oligonucleotideprobe. The presence of specific hybridization of the probe to theamplified SETX is then detected. Specific hybridization of anallele-specific oligonucleotide probe to DNA from the individual isindicative of the presence of a polymorphism of interest.

In another embodiment of the invention, fluorescence resonance energytransfer (FRET) can be used to detect the presence of a polymorphism ofinterest. FRET is the process of a distance-dependent excited stateinteraction in which the emission of one fluorescent molecule is coupledto the excitation of another. A typical acceptor and donor pair forresonance energy transfer consists of 4-[[4-(dimethylamino) phenyl]azo]benzoic acid (DABCYL) and 5-[(2-aminoethylamino]naphthalene sulfonicacid (EDANS). EDANS is excited by illumination with 336 nm light, andemits a photon with wavelength 490 nxn. If a DABCYL moiety is locatedwithin 20 angstroms of the EDANS, this photon will be efficientlyabsorbed. DABCYL and MANS will be attached to two differentoligonucleotide probes designed to hybridize head-to-tail to nucleicacid adjacent to and/or overlapping the site of one of the mutations ofinterest. Melting curve analysis is then applied: cycles ofdenaturation, cooling, and re-heating are applied to a test sample mixedwith the oligonucleotide probes, and the fluorescence is continuouslymonitored to detect a decrease in DABCYL fluorescence or an increase inEDANS fluorescence (loss of quenching). While the two probes remainhybridized adjacent to one another, FRET will be very efficient.Physical separation of the oligonucleotide probes results in inefficientFRET, as the two dyes are no longer be in close proximity. The presenceor absence of a mutation of interest can be assessed by comparing thefluorescence intensity profile obtained from the test sample, tofluorescence intensity profiles of control samples comprising knownmutations of interest in the SETX gene.

In another embodiment, arrays of oligonucleotide probes that arecomplementary to target nucleic acid sequence segments from anindividual, can be used to identify polymorphisms of interest. Forexample, in one embodiment, an oligonucleotide array can be used.Oligonucleotide arrays typically comprise a plurality of differentoligonucleotide probes that are coupled to a surface of a substrate indifferent known locations. These oligonucleotide arrays, also describedas “Genechips™,” have been generally described in the art, for example,U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and92/10092. These arrays can generally be produced using mechanicalsynthesis methods or light directed synthesis methods which incorporatea combination of photolithographic methods and solid phaseoligonucleotide synthesis methods. See Fodor et al., Science,251:767-777 (1991), Pirrung et al., U.S. Pat. No. 5,143,854 (see alsoPCT Application No. WO 90/15070) and Fodor et al., PCT Publication No.WO 92/10092 and U.S. Pat. No. 5,424,186, the entire teachings of each ofwhich are incorporated by reference herein. Techniques for the synthesisof these arrays using mechanical synthesis methods are described in,e.g., U.S. Pat. No. 5,384,261, the entire teachings of which areincorporated by reference herein.

Once an oligonucleotide array is prepared, a nucleic acid of interest ishybridized with the array and scanned for polymorphisms. Hybridizationand scanning are generally carried out by methods described herein andalso in, e.g., Published PCT Application Nos. WO 92/10092 and WO95/11995, and U.S. Pat. No. 5,424,186, the entire teachings of which areincorporated by reference herein. In brief, a target nucleic acidsequence which includes one or more previously identified polymorphicmarkers is amplified by well known amplification techniques, e.g., PCR.Typically, this involves the use of primer sequences that arecomplementary to the two strands of the target sequence both upstreamand downstream from the polymorphism. Asymmetric PCR techniques may alsobe used. Amplified target, generally incorporating a label, is thenhybridized with the array under appropriate conditions. Upon completionof hybridization and washing of the array, the array is scanned todetermine the position on the array to which the target sequencehybridizes. The hybridization data obtained from the scan is typicallyin the form of fluorescence intensities as a function of location on thearray.

Although primarily described in terms of a single detection block, e.g.,for detection of a single polymorphism, arrays can include multipledetection blocks, and thus be capable of analyzing multiple, specificpolymorphisms. In alternate arrangements, it will generally beunderstood that detection blocks may be grouped within a single array orin multiple, separate arrays so that varying, optimal conditions may beused during the hybridization of the target to the array. For example,it may often be desirable to provide for the detection of thosepolymorphisms that fall within G-C rich stretches of a genomic sequence,separately from those falling in A-T rich segments. This allows for theseparate optimization of hybridization conditions for each situation.

Additional description of use of oligonucleotide arrays for detection ofpolymorphisms can be found, for example, in U.S. Pat. Nos. 5,858,659 and5,837,832, the entire teachings of which are incorporated by referenceherein.

Other methods of nucleic acid analysis can be used to detectpolymorphisms of interest. Representative methods include direct manualsequencing (Church and Gilbert, (1988), Proc. Natl. Acad. Sci. USA81:1991-1995; Sanger, F. et al. (1977) Proc. Natl. Acad. Sci.74:5463-5467; Beavis et al. U.S. Pat. No. 5,288,644); automatedfluorescent sequencing; single-stranded conformation polymorphism assays(SSCP); clamped denaturing gel electrophoresis (CDGE); denaturinggradient gel electrophoresis (DGGE) (Sheffield, V. C. et al. (19891)Proc. Natl. Acad. Sci. USA 86:232-236), mobility shift analysis (Orita,M. et al. (1989) Proc. Natl. Acad. Sci. USA 86:2766-2770; Rosenbaum andReissner (1987) Biophys. Chem., 265:1275; Keen et al. (1991) TrendsGenet., 7:5; restriction enzyme analysis (Flavell et al. (1978) Cell15:25; Geever, et al. (1981) Proc. Natl. Acad. Sci. USA 78:5081);heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et al.(1985) Proc. Natl. Acad. Sci. USA 85:4397-4401); RNase protection assays(Myers, R. M. et al. (1985) Science 230:1242); use of polypeptides whichrecognize nucleotide mismatches, such as E. coli mutS protein (See, forexample, U.S. Pat. No. 5,459,039); Luminex xMAP™ technology; and/orallele-specific PCR, for example.

These methods can be used to identify the presence of one or moremutations of interest in the SETX gene as described herein. For example,in certain embodiments, the methods can be used to assess both the firstand the second alleles of the SETX gene of an individual for thepresence of one or more polymorphisms of interest. The terms, “first”and “second” alleles are -arbitrarily applied to the two alleles; thatis, either allele may be designated as the “first” allele, and the otherallele is then designated as the “second” allele.

In one particular embodiment of the invention, the methods of assessinga test sample for the presence or absence of a mutation in the SETXgene, as described above, are used to assess an individual for thepresence or absence of a genetic polymorphism associated with AOA2. Thepresence of at least one of the mutations of interest (e.g., a singlebase insertion of T between nucleotides 479-480; a 4 base deletion ofnucleotides 4633-4636; a 2 base deletion of nucleotides 6114-6115; asingle base transition C→T at nucleotide 6292; a 4 base deletion ofnucleotides 369-372; a 2 base insertion of AT between nucleotides2747-2748; a single base transition C→T at nucleotide 4234; a singlebase transition C→T at nucleotide 4816; a 6 base deletion of nucleotides4873-4878 accompanied by an insertion of GG at the same location; asingle base insertion of G between nucleotides 4891-4892; a 2 baseinsertion of CA between nucleotides 5301-5302; a 4 base deletion ofnucleotides 5308-5311; a 2 base deletion of nucleotides 5591-5592; asingle base deletion of nucleotide 5958; a single base insertion of Abetween nucleotides 6422-6423; or a four base deletion of nucleotides6848-6851) is indicative of the presence of a genetic polymorphismassociated with AOA2.

In another embodiment of the invention, the methods of assessing a testsample for the presence or absence of a mutation in the SETX gene, asdescribed above, are used to diagnose AOA2 in an individual. BecauseAOA2 is a recessive cerebellar ataxia, identification of a mutationassociated with AOA2 in each allele of the SETX gene of an individual isnecessary for diagnosis of AOA2. The two alleles may have the samemutation present, or may have different mutations; furthermore, morethan one mutation may be found in one or both alleles. In these methods,at least one polymorphism of interest (a single base insertion of Tbetween nucleotides 479-480; a 4 base deletion of nucleotides 4633-4636;a 2 base deletion of nucleotides 6114-6115; a single base transition C→Tat nucleotide 6292; a 4 base deletion of nucleotides 369-372; a 2 baseinsertion of AT between nucleotides 2747-2748; a single base transitionC→T at nucleotide 4234; a single base transition C→T at nucleotide 4816;a 6 base deletion of nucleotides 4873-4878 accompanied by an insertionof GG at the same location; a single base insertion of G betweennucleotides 4891-4892; a 2 base insertion of CA between nucleotides5301-5302; a 4 base deletion of nucleotides 5308-5311; a 2 base deletionof nucleotides 5591-5592; a single base deletion of nucleotide 5958; asingle base insertion of A between nucleotides 6422-6423; or a four basedeletion of nucleotides 6848-6851) is found in at least one of the twoalleles of the SETX gene (the “first” allele). In addition, at least onemutation associated with AOA2 is present in the other allele of the SETXgene (the “second” allele). The mutation associated with AOA2 that ispresent in the second allele can be a polymorphism of interest asdescribed herein; alternatively, the mutation associated with AOA2 thatis present in the second allele can be a mutation in SETX that has beenpreviously associated with AOA2, such as the mutations set forth inTable 1, below.

TABLE 1 Mutations in SETX Previously Associated with AOA2 Gene cDNA ntLocation Number nt Change Codon aa Change Outcome Reference Exon 3 8 C8T3 T3I Missense 2 Exon 4 193 G193A 65 E65K Missense 3 Exon 8 879 879delT292 fs after 292 Frameshift 1 Exon 8 915 G915T 305 W305C Missense 1 Exon8 994 C994T 332 R332W Missense 1 Exon 10 1166 T1166C 389 L389S Missense2 Exon 10 1238 C1238T 413 P413L Missense 1 Exon 10 2332 C2332T 788 R778XNonsense 1 Exon 10 2602 C2602T 868 Q868X Nonsense 1 Exon 10 2622-26252622-2625delAGTT 874 fs after 874 Frameshift 1 Exon 10 2966-29702966-2970delGGAAA 988 fs after 988 Frameshift 1 Exon 10 4087 C4087T 1363R1363X Nonsense 1 Exon 10 4321 C4321T 1441 Q1441X Nonsense 1 Exon 105070 5070insT 1690 fs after 1690 Frameshift 1 Exon 10 5249 5249insT 1750fs after 1750 Frameshift 1 Exon 10 5264 5264delC 1754 fs after 1754Frameshift 1 Exon 10 5267 T5267C 1756 F1756S Missense 1 Exon 14 5927T5927G 1976 L1276R Missense 3 Exon 19 6407 G6407A 2136 R2136H Missense 2Exon 20 6638 C6638T 2213 P2213L Missense 1 1 Moreira, M C, et al.Senataxin, the ortholog of a yeast RNA helicase, is mutant inataxia-ocular apraxia 2. Nature Genetics 2004. March 36(3): 225-7. 2Chen, Y Z, et al. DNA/RNA helicase gene mutations in a form of JuvenileAmyotrophic Lateral Sclerosis (ALS4). Am. J. Hum. Genet. 2004 (74):1128-35. 3 Duquette, A, et al. Mutations in Senataxin responsible forQuebec cluster of Ataxia with Neuropathy. Ann. Neurol. 2005. March57(3): 408-14.

It should be noted that while one or both of the alleles may have amutation as set forth in Table 1, the methods of the invention requirethat one or more of the polymorphisms of interest be present in at leastone of the alleles, or in both of the alleles, for diagnosis of AOA2 bythe methods of the invention.

In a further embodiment of the invention, the methods of assessing atest sample for the presence or absence of a mutation in the SETX gene,as described above, are used to diagnose carrier status of an individualfor AOA2. The term, “carrier status,” indicates that the individualcarries mutation of interest in a single allele of the SETX gene, and nomutations associated with AOA2 in the second allele, and thus isconsidered a carrier for this recessive cerebellar ataxia. In thesemethods, at least one polymorphism of interest (a single base insertionof T between nucleotides 479-480; a 4 base deletion of nucleotides4633-4636; a 2 base deletion of nucleotides 6114-6115; a single basetransition C→T at nucleotide 6292; a 4 base deletion of nucleotides369-372; a 2 base insertion of AT between nucleotides 2747-2748; asingle base transition C→T at nucleotide 4234; a single base transitionC→T at nucleotide 4816; a 6 base deletion of nucleotides 4873-4878accompanied by an insertion of GG at the same location; a single baseinsertion of G between nucleotides 4891-4892; a 2 base insertion of CAbetween nucleotides 5301-5302; a 4 base deletion of nucleotides5308-5311; a 2 base deletion of nucleotides 5591-5592; a single basedeletion of nucleotide 5958; a single base insertion of A betweennucleotides 6422-6423; or a four base deletion of nucleotides 6848-6851)is found in only a first allele of the two alleles of the SETX gene (inthe “first” allele). In addition, no mutations associated with AOA2 arefound in the second allele of the SETX gene.

It is noted that benign polymorphisms may be present in either or bothalleles of the SETX gene.

The present invention also pertains to kits (e.g., reagent kits) usefulin the methods of the invention. Such kits comprise components useful inany of the methods described herein, including for example,hybridization probes or primers (e.g., labeled probes or primers),reagents for detection of labeled molecules, restriction enzymes (e.g.,for RFLP analysis), allele-specific oligonucleotides, means foramplification of nucleic acids comprising SETX or a fragment of SETX, ormeans for analyzing the nucleic acid sequence of SETX. For example, inone embodiment, the kit comprises components useful for analysis ofmutations of interest using microsphere-based technology such as LuminexxMAP™ technology. In a preferred embodiment of the invention, the kitcomprises components for detecting one or more of the mutations ofinterest (a single base insertion of T between nucleotides 479-480; a 4base deletion of nucleotides 4633-4636; a 2 base deletion of nucleotides6114-6115; a single base transition C→T at nucleotide 6292; a 4 basedeletion of nucleotides 369-372; a 2 base insertion of AT betweennucleotides 2747-2748; a single base transition C→T at nucleotide 4234;a single base transition C→T at nucleotide 4816; a 6 base deletion ofnucleotides 4873-4878 accompanied by an insertion of GG at the samelocation; a single base insertion of G between nucleotides 4891-4892; a2 base insertion of CA between nucleotides 5301-5302; a 4 base deletionof nucleotides 5308-5311; a 2 base deletion of nucleotides 5591-5592; asingle base deletion of nucleotide 5958; a single base insertion of Abetween nucleotides 6422-6423; or a four base deletion of nucleotides6848-6851).

The present invention is explained in greater detail in the followingnon-limiting examples.

EXAMPLE 1 Identification of Mutations in SETX Gene Associated with AOA2

Patients

Patients assessed for the presence of mutations of interest in the SETXgene included individuals who had previously been diagnosed with ataxia.Characteristics of AOA2 demonstrated by patients generally included oneor more of the following: clinical onset between 10 and 22 years of age;severe gait disorder with mildly affected limb and trunk movement;opthalmological symptoms including variably present oculomotor apraxia,disorder smooth pursuit and saccade palsy; peripheral neuropathyincluding sensory-motor and axonal loss; elevated alpha-fetoprotein(AFP) levels; absent tendon reflexes in legs.

Mutation Screening

Whole blood specimens were obtained from patients, and DNA was extractedusing the Puregene Extraction Method (Gentra Systems Inc., Minneapolis,Minn.). Polymerase chain reaction (PCR) was used to amplify genomic DNA;the PCR products were then purified, and cycle sequencing was performed.An ABI Prism® 3730 Genetic Analyzer was used for automated sequencing.

The SETX gene is comprised of 27 exons through 92 kilobases, having an8,031 base pair coding region that begins within exon 3 and continues toexon 26. To assess for mutations of interest in the SETX gene, all ofthe SETX coding exons were sequenced. To sequence the SETX coding exons,the gene was broken down into amplicons ranging in size from 311 basepairs to 549 base pairs, in order to allow high quality sequencing ofeach exon and splice junction with a single primer. Smaller exons (exons3-9 and 11-25) were amplified using individual exons (including splicejunctions) as the amplicons, and exons 10 (4,175 base pairs) and 26 (746base pairs) were amplified using multiple amplicons. Mutations wereverified by sequencing the opposite strand. Primers for amplicons weredesigned not to overlap with k known deletions or insertions, tominimize the possibility that DNA variants affecting PCR priming siteswould result in allele dropout and false normal results. Referencecontrols included genomic DNA having an identified SETX sequencealteration (positive control), and/or genomic DNA having no SETXalterations (normal controls). Sequences were compared to the SETX mRNAsequence, shown in SEQ ID NO:1.

Results

Several mutations in the SETX gene associated with AOA2 were identified.They are set forth in Table 2, below.

TABLE 2 Mutations of Interest in the SETX Gene, Associated With AOA2cDNA nt Number nt Change Outcome 369-372 4 bp Deletion Frame Shift2747-2748 2 bp Insertion of AT Frame Shift 4234 Transition C > TGlutamine > Amber (Homozygous) (Stop codon) 4816 Transition C > TArginine > OPA (Stop codon) 4873-4878 4873-4878del6insGG Frame Shift4891-4892 1 bp Insertion of G Frame Shift 5301-5302 2 bp Insertion of CAFrame Shift (Homozygous) 5308-5311 4 bp Deletion Frame Shift 5591-5592 2bp Deletion of AA Frame Shift (Homozygous) 5958 1 bp Deletion of G FrameShift 6292 Transition C > T Arginine > TGA (Stop codon) 6422-6423 1 bpInsertion of A Frame Shift (Homozygous) 6848-6851 4 bp Deletion FrameShift 479-480 1 bp insertion of T Frame Shift 4633-4636 4 bp deletion ofAGTG Frame Shift (Homozygous) 6114-6115 2 bp Deletion of TG Frame ShiftOf these mutations, five were homozygous; the presence of the homozygousmutation, in combination with AOA2 symptoms, indicated that thesemutations were associated with the presence of AOA2. The remainder ofthe mutations were heterozygous, and were associated with AOA2 becausethe individuals having those mutations in one allele were symptomaticfor AOA2 and also had a second mutation associated with AOA2 present inthe second allele.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1. A method of assessing a human individual for the presence of agenetic polymorphism associated with ataxia-ocular apraxia 2 (AOA2), themethod comprising a) assessing a test sample from the individual for thepresence of a mutation of interest in the senataxin (SETX) gene, whereinthe mutation of interest is a 4 base deletion of nucleotides 369-372 b)detecting the presence of the 4 base deletion of nucleotides 369-372wherein the nucleotide numbering is as shown in SEQ ID NO: 1, andwherein the presence of the mutation of interest is indicative of thepresence of a genetic polymorphism associated with ataxia-ocular apraxia2.
 2. The method of claim 1, wherein the test sample from the individualcomprises genomic DNA.
 3. The method of claim 2, wherein the genomic DNAcomprises chromosome 9 or a fragment thereof comprising 9q34.
 4. Themethod of claim 1, wherein assessing the test sample comprisesamplifying all or a fragment of the senataxin gene.
 5. The method ofclaim 1, wherein assessing the test sample comprises direct sequenceanalysis.
 6. The method of claim 1, further comprising assessing thetest sample from the individual for the presence of at least one furthermutation of interest in the senataxin (SLTX) gene, wherein the furthermutation of interest is: a 2 base insertion of AT between nucleotides2747-2748; a single base transition C→T at nucleotide 4234; a singlebase transition C→T at nucleotide 4816; a 6 base deletion of nucleotides4873-4878 accompanied by an insertion of GG at the same location; asingle base insertion of G between nucleotides 4891-4892; a 2 baseinsertion of CA between nucleotides 5301-5302; a 4 base deletion ofnucleotides 5308-5311; a 2 base deletion of nucleotides 5591-5592; asingle base deletion of nucleotide 5958; a single base insertion of Abetween nucleotides 6422-6423; a single base transition C→T atnucleotide 6292; a four base deletion of nucleotides 6848-6851; a singlebase insertion of T between nucleotides 479-480; a 4 base deletion ofnucleotides 4633-4636; or a 2 base deletion of nucleotides 6114-6115. 7.A method of diagnosing ataxia-ocular apraxia 2 (AOA2) in a humanindividual with ataxia, a) assessing a test sample from the individualfor the presence of a mutation of interest in a first allele of thesenataxin (SETX) gene of the individual, wherein the mutation ofinterest is a 4 base deletion of nucleotides 369-372 b) diagnosing thehuman as having AOA2 when the 4 base deletion of nucleotides 369-372 isdetected in the first allele and any mutation associated with AOA2 isalso present in a second allele of the SETX gene wherein the nucleotidenumbering is as shown in SEQ ID NO:
 1. 8. The method of claim 7, whereinthe test sample from the individual comprises genomic DNA.
 9. The methodof claim 8, wherein the genomic DNA comprises chromosome 9 or a fragmentthereof comprising 9q34.
 10. The method of claim 7, wherein assessingthe test sample comprises amplifying all or a fragment of the senataxingene.
 11. The method of claim 7, wherein assessing the test samplecomprises direct sequence analysis.
 12. The method of claim 7, furthercomprising assessing a test sample from the individual for the presenceof at least one further mutation of interest in a first allele of thesenataxin (SETX) gene of the individual, wherein the further mutation ofinterest is: a 2 base insertion of AT between nucleotides 2747-2748; asingle base transition C→T at nucleotide 4234; a single base transitionC→T at nucleotide 4816; a 6 base deletion of nucleotides 4873-4878accompanied by an insertion of GG at the same location; a single baseinsertion of G between nucleotides 4891-4892; a 2 base insertion of CAbetween nucleotides 5301-5302; a 4 base deletion of nucleotides5308-5311; a 2 base deletion of nucleotides 5591-5592; a single basedeletion of nucleotide 5958; a single base insertion of A betweennucleotides 6422-6423; a single base transition C→T at nucleotide 6292;a four base deletion of nucleotides 6848-6851; a single base insertionof T between nucleotides 479-480; a 4 base deletion of nucleotides4633-4636; or a 2 base deletion of nucleotides 6114-6115.
 13. The methodof claim 12, wherein both the first and the second alleles of thesenataxin (SETX) gene comprise a mutation of interest selected from thegroup consisting of: a) a 4 base deletion of nucleotides 369-372; b) a 2base insertion of AT between nucleotides 2747-2748; c) a single basetransition C→T at nucleotide 4234; d) a single base transition C→T atnucleotide 4816; e) a 6 base deletion of nucleotides 4873-4878accompanied by an insertion of GG at the same location; f) a single baseinsertion of G between nucleotides 4891-4892; g) a 2 base insertion ofCA between nucleotides 5301-5302; h) a 4 base deletion of nucleotides5308-5311; i) a 2 base deletion of nucleotides 5591-5592; j) a singlebase deletion of nucleotide 5958; k) a single base insertion of Abetween nucleotides 6422-6423; l) a single base transition C→T atnucleotide 6292; m) a four base deletion of nucleotides 6848-6851; n) asingle base insert ion of T between nucleotides 479-480; o) a 4 basedeletion of nucleotides 4633-4636; and p) a 2 base deletion ofnucleotides 6114-6115.
 14. A method of assessing a human individual forcarrier status for ataxia-ocular apraxia 2 (AOA2), the method comprisinga) assessing a test sample from the individual for the presence of amutation of interest in first and second alleles of the senataxin (SETX)gene of the individual, wherein the mutation of interest is a 4 basedeletion of nucleotides 369-372 b) detecting the presence of the 4 basedeletion of nucleotides 369-372 in the first allele of the senataxingene, and the absence of any mutation associated with ataxia-ocularapraxia 2 in the second allele of the senataxin gene is indicative ofcarrier status for ataxia-ocular apraxia 2 wherein the nucleotidenumbering is as shown in SEQ ID NO:
 1. 15. The method of claim 14,wherein the test sample from the individual comprises genomic DNA. 16.The method of claim 15, wherein the genomic DNA comprises chromosome 9or a fragment thereof comprising 9q34.
 17. The method of claim 14,wherein assessing the test sample comprises amplifying all or a fragmentof the senataxin gene.
 18. The method of claim 14, wherein assessing thetest sample comprises direct sequence analysis.
 19. The method of claim14, further comprising assessing a test sample from the individual forthe presence of at least one further mutation of interest in first andsecond alleles of the senataxin (SETX) gene of the individual, whereinthe further mutation of interest is: a 2 base insertion of AT betweennucleotides 2747-2748; a single base transition C→T at nucleotide 4234;a single base transition C→T at nucleotide 4816; a 6 base deletion ofnucleotides 4873-4878 accompanied by an insertion of GG at the samelocation; a single base insertion of G between nucleotides 4891-4892; a2 base insertion of CA between nucleotides 5301-5302; a 4 base deletionof nucleotides 5308-5311; a 2 base deletion of nucleotides 5591-5592; asingle base deletion of nucleotide 5958; a single base insertion of Abetween nucleotides 6422-6423; a single base transition C→T atnucleotide 6292; a four base deletion of nucleotides 6848-6851; a singlebase insertion of T between nucleotides 479-480; a 4 base deletion ofnucleotides 4633-4636; or a 2 base deletion of nucleotides 6114-6115.